Marys Medicine

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology

Student name: Tran Thai My Duyen – BTBTIU10019
Supervisor: Dr. Nguyen ThaoTrang
In my first word, I wish to thank my parents for their love, unconditional support and encouragement throughout my thesis. They help me realize my own potential over the years. I would like to express my gratitude to lecturers and academic staffs in the School of Biotechnology for providing me a great working environment during the completion of my thesis work. Next, I would like to express my deepest appreciation to my supervisor at the school of Biotechnology - International University, Dr. Nguyen Thao Trang, who gave me huge support all along. I really admire her wide knowledge and skills in scientific area. During my thesis period, not only she passionately taught me valuable academic knowledge but she also taught me lots of precious things beside science. I would like to say that having opportunity to be under her supervision has been my highest pleasure. Thanks to her heartfelt advices and supports during my thesis registration and completing report. Last but not least, a very special thanks goes to Ms. Tran Thi Quynh Dao, Ms. Nguyen Thi Xuan Huong, who has spent countless hours in the lab explaining and instructing me how to carry out the experiments. In addition, I would like to thank all the other officers at Applied Chemistry Laboratory and many third-year students, namely, To VinhTrieu, Nguyen QuanTrinh, Dao Ngoc Phuong Uyen at International University for enthusiastically supporting me during my thesis. EFFECT OF CHOLESTEROL ON THE PARTITIONING OF
Duyen T.M. Trana, Trieu V. To, Trang T. Nguyenb aSchool of Biotechnology, International University – Vietnam National University in HCMC bCorresponding author's email address: ABSTRACT
In this study, the effect of cholesterol on the partitioning of amitriptyline, a tricyclic antidepressant, into lipid bilayers composed of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was examined using second derivative spectrophotometric method. As the results revealed, amitriptyline preferred to partition into the unsaturated DOPC followed by the mixed chain (SOPC) and the saturated (DSPC). The presence of 28 mol% cholesterol facilitated the partitioning of amitriptyline into the saturated and mixed chain lipids (DSPC and SOPC) but decreased the drug partitioning into the unsaturated lipid (DOPC). The study showed a significant role of cholesterol on the partitioning of a drug into the lipid membranes. Keywords: Amitriptyline, Cholesterol, Liposomes, Second Derivative Spectrophotometer
The therapeutic and toxic effects of drugs are strongly influenced by their lipid affinity, and the study of drug-lipidmembrane interaction is of importance in drug absorption, distribution, metabolism and elimination phenomena, as well as in assessing toxic or therapeutic effects and bioaccumulation. Lipid membranes contain several hundred types of lipids with different headgroups and acyl chain compositions whose properties such as charge state and packing density will influence drug partitioning. The major component of membrane lipids is glycerophospholipids which are comprised of a polar headgroup and two nonpolar acyl chains as a tail. The most popular headgroup is phosphatidylcholines (PC) which are electrically neutral incorporate choline as a headgroup. The two acyl chains may be saturated, unsaturated or one chain saturated and the other unsaturated. As stated above, the difference in the unsaturation degree results in difference in the lipid fluidity and packing density, and thus will affect the partitioning of the drug to the lipid membrane. It has been found that one of the most important components of cell membranes which influences on the cell membranes' activity is cholesterol. Cholesterol is a modified steroid and plays an essential structural component of cell membranes that is required to regulate membrane permeability and fluidity by changing their ordering, available area and formation of domains of composition. At the molecular level, the most pronounced and easily identified effects of cholesterol are the so-called ordering and condensing effects on membrane lipids; cholesterol has a dual nature - it promotes ordering and rigidity of the lipids in the liquid state, while it's effects are the opposite on the gel state lipids. There are several studies about drug-lipid membrane interactions and distribution of drug into lipid membranes depending on the saturation of lipid alkyl chains. In addition, many studies have revealed the general interaction between cholesterol and phospholipid bilayer (e.g., cholesterol interacts with all of the lipid in bilayer membrane, cholesterol-induced fluid membrane domains, complex behavior phosphocholine/cholesterol). The effect of cholesterol on the structure of lipid membrane has been studied more clearly (e,g., effect of cholesterol on phosphatidylcholine bilayer polar region; relationships between hydrophobic thickness, acyl-chain orientation order of lipid membrane and cholesterol; effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers; importance of double-bond position on interplay of unsaturated phospholipids and cholesterol in membrane). However,it is still unclear how cholesterol affects the partition of a drug into lipid membranes. Because cholesterol fluidizes the lipid membranes if lipids are in the gel-state whereas the lipid bilayers in the liquid-crystalline state become more ordered with the presence of cholesterol. Moreover, in the presence of cholesterol, this involves one assumption that cholesterol occupies more space that prevents the drug from partitioning into the lipid membrane. Therefore, whether cholesterol enhances or impedes the partitioning of drugs into the lipids with different unsaturation degree should be examined. Amitriptyline is a type of medicine called a tricyclic antidepressant (TCA) which acts on nerve cells in the brain. When depression occurs, there may be a decreased amount of serotonin and noradrenaline released from nerve cells in the brain. Amitriptyline works by preventing serotonin and noradrenaline from being reabsorbed back into the nerve cells in the brain. This helps prolong the mood lightening effect of any released noradrenaline and serotonin. In this way, amitriptyline helps relieve depression. Due to the fact that amitriptyline inhibits the membrane pump mechanism which responsibles for the uptake of noradrenaline and serotonin in adrenergic and serotonergic neurons, it has been generally believed that drug inhibition ability correlates with its mechanism of partition into lipid membranes. In this study, the effect of cholesterol on the partitioning of amitriptyline into lipid membranes was examined. The partitioning of a drug into lipid membranes can be expressed through a partition coefficient (Kp). Kp is an indicator of the distribution of a drug between lipid and aqueous phases. It is a key parameter in drug design as the absorption, distribution, metabolism as well as toxicity and therapeutic effects of a drug involve its passage across lipid membranes. Therefore, the effect of cholesterol on the lipid membrane partitioning of the drug can be evaluated by the Kp. The coefficient (Kp) of amitriptyline into the lipid membranes with and without cholesterol (28 mol%) was determined by using stable immobilized unilamellar liposomes which are model mammalian cell membranes. The partition coefficient of amitriptyline were examined in 3 lipids which are different in the unsaturation glycero-3-phosphocholine (SOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). All these three lipids are glycerophospholipids, which are comprised of 2 acyl chains and a polar head group. DSPC has two saturated acyl chains while DOPC is composed of two unsaturated chains. SOPC is the mixed chain lipid with one chain saturated and the other unsaturated. The chemical structures of DOPC, SOPC and DSPC were shown in Figure 1. The varying unsaturation degree leads to the difference in lipid fluidity and packing density, therefore affects the partitioning of the drug into the lipid membranes. 1) Materials:
phosphocholine (SOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were bought from Avanti polar lipids (USA). Amitriptyline was purchased from Sigma Aldrich (USA). Cholesterol (99 +% purity, Sigma Chemical Co.). Nanopure

water, distilled from NanopureTM system with impedance of 18.2 MΩ-cm, was used to prepare all solutes during the experiments. All liquid suspensions were made with PBS buffer solution (50 mM Na2HPO4.2H2O and 100 mMNaCl (Merck, Germany) at pH 7.4). Figure 1:Chemical structures of DOPC, SOPC and DSPC.
Figure 2: Structure of Cholesterol, Amitriptyline (Sigma Aldrich, USA) and
Unilamellar liposome (the liposome structure was taken from FAO Corporate
Document Repository).
2) Liposome and drug/liposome preparation
The pure lipids (DOPC, SOPC and DSPC) and lipids containing 28 mol% cholesterol were thoroughly mixed in chloroform and then evaporated to dryness under the stream of nitrogen. The dried lipid film was left under vacuum overnight to remove all traces of the organic solvent and then stored at -20 oC until used. In order to prepare liposomes, PBS buffer was added into the dried lipid vial and the mixture was vortexed to produce multicellular liposomes (MLVs). After that, MLVs were frozen and thawed by repeating 5 times a cycle of freezing the liposomes in -20 oC and then thawing in a water bath at 60 oC. Next, the lipid suspensions were extruded 30 times through polycarbonate filters with a pore size of 0.1 m to produce unilamellar vesicles (LUVs). During extrusion, the lipid solutions were kept at the temperature at least 10 oC higher than the phase transition temperature for each lipid, room temperature for DOPC and SOPC and 65 oC for DSPC. Sample solutions were prepared by mixing a known volume of drug and suitable vesicle suspensions. The lipid stock suspension was diluted to prepare a set of suspension with different lipid concentration (range from 0 to 0.25 mM), in which the drug concentration was kept constant at 0.0225 mM. A set of blank suspensions (corresponding reference solutions) were prepared identically but without amitriptyline for each assay. All suspensions were vortexed for 30 seconds and then incubated at 37 oC for at least 30 minutes before being measured. 3) UV-Vis measurement
The absorption spectra of all suspensions were collected using Agilent Cary 60 UV- Vis spectrophotometer with the spectral window from 190nm to 300 nm and equipped with a constant-temperature cell holder. The absorption spectra of sample suspensions were obtained by measuring against the corresponding reference suspension which had the same composition but without amitriptyline. All sample solutions were measured at 37 oC in a microcell cuvette with a chamber volume of 4) Determination of partition coefficients
Partition coefficients were determined by the derivative spectrophotometry. This technique is based on the evaluation of the discrete spectral variations presented by the drug in the presence of increasing lipid concentrations. The liposome/buffer patition coefficient is defined as the ratio between the concentration of membrane- bound drug in lipid phase and the concentration of free drug in buffer phase. This relation can be expressed as: Where Ct: drug molar concentration Cm: drug in lipid concentration Cw: drug in aqueous media concentration [lipid]: lipid molar concentration [water]: water molar concentration (55.3 M at 37 oC) According to the Beer-Lambert law, absorbance is directly proportional to concentration, at a specific wavelength, A = εmCm + εwCw Where εm: drug extinction coefficient in lipid bilayer εw: drug extinction coefficient in water Let ∆A is the difference between absorption in the presence and absence of liposomes and related to portioning coefficient (Kp value) by the following equation: Similar to absorbance, derivative intensity is proportional to the solute concentration. Denoting (dnA/dnλ) by D. From equation (1) and (2), relation between ∆D and Kp could be expressed by the following equation: Where ∆D is differential absorption of drug in lipid phase at a high concentration of lipid, that is, when 100 % of drug binds to liposomes, ∆D reaches its maximum value ∆Dmax, where ∆Dmax = εCt. The values of Kp and ∆Dmax were calculated from the experimental values of molar concentration of lipid and ∆D by applying a non- linear least-squares method. The derivative spectra were calculated using OriginPro 9.0 software (OriginLab, Northampton, MA) that involved the Savitzky- Golay method, in which the second-order polynomial and 17 window points Employing the derivative spectrophotometry method, light scattering from lipid vesicles was eliminated before measuring patition coefficient. The Kp values were calculated by fitting experimental data of ∆D and molar concentration of lipid to equation (3). Applying maximum-peak method for heterogeneous samples in order to increase reproducibility and signal-to-noise ratio. ∆D values were collected at λmax of the absorption spectra. Because light scattering as a source of additional noise in absorption measurements, ∆D values used were obtained atthe wavelength (λmax) where maximum absorbance of amitriptyline was occurred. RESULTS AND DISCUSSION
1) Absorption spectra
a. Absorption spectra of amitriptyline in pure lipids The absorption spectra of amitriptyline at a concentration of 0.0225 mM in the presence of various amounts of lipid vesicles containing DOPC, SOPC and DSPC were depicted in Figure 3. It is important to point out that the concentration of amitriptyline used in the study was obeyed Beer's Law for absorption spectra. The curves (2-8) in Figure 3 were obtained by subtracting the absorption spectrum of lipid without amitriptyline (the blank) from absorption spectrum of lipid with amitriptyline recorded at the same lipid concentration. When increasing the lipid concentration of DOPC, SOPC and DSPC, the maximum absorbance at the wavelength of 209 nm decreased and the wavelength of the maxima showed bathochromic shifts – shifts to longer wavelength. Similar shifts in absorption spectra have been previously observed for chlorpromazine, promazine and methochlorpromazine. This demonstrated that amitriptyline partitioned into the LUVs, ie., the environment surrounding amitriptyline became less polar as amitriptyline partitioned from the aqueous phase to the lipid phase. Figure 3: Absorption spectra of 0.0225 mM amitriptyline in PBS buffer solution (pH
7.4, 37 oC) containing various amounts of LUVs (DOPC, SOPC, DSPC, respectively). Lipid vesicles concentrations (mM): (1) 0; (2) 0.025; (3) 0.05; (4) 0.075; (5) 0.1; (6) 0.15; (7) 0.2; (8) 0.25. b. Absorption spectra of amitriptyline in lipids containing 28 mol% cholesterol The absorption spectra of amitriptyline at a concentration of 0.0225 mM in the lipid vesicles of DOPC, SOPC and DSPC containing 28 mol% cholesterol were shown in In the presence of 28 mol% cholesterol, the absorption spectra of amitriptyline in the three lipids (DOPC, SOPC and DSPC) were similar to those in the pure lipids. The absorbance of amitriptyline in the three DOPC, SOPC and DSPC lipids also decreased and the wavelengths of the maxima shifted to the right. The background signals presented by the lipid solutions in the ultraviolet region which could not be eliminated by zero-order spectra. Applying higher orders of derivative, particularly, second-order derivative could eliminate baseline shifts, since scattering by lipid had a negligible effect on the second derivative. Moreover, second derivative spectrophotometry increased the accuracy of quantification because spectral details were enhanced and overlapping bands were separated. DOPC + CHOLESTEROL
Figure 4: Absorption spectra of 0.0225 mM amitriptyline in PBS buffer solution (pH
7.4, 37 oC) containing various amounts of LUVs/Cholesterolvescicles (lipid DOPC, SOPC, DSPC, respectively). The lipid vesicles concentrations (mM): (1) 0; (2) 0.025; (3) 0.05; (4) 0.075; (5) 0.1; (6) 0.15; (7) 0.2; (8) 0.25. 2) Second derivative spectra of absorption
a. Second derivative spectra of absorptionin the pure lipids Figure 5: Second derivative spectra of amitriptyline calculated from the absorption
spectra in Figure 3. The second derivative absorption spectra of amitriptyline in different lipid concentrations were shown in Figure 5. As can be observed, the interference caused by the presence of liposomes was completely eliminated with the second derivative. The second derivative absorbance minima increased in intensity and shifted toward higher wavelengths. b. Second derivative spectra of absorbance in the lipids containing 28 mol% cholesterol Second derivative absorption spectra of amitriptyline in the lipids containing 28 mol% cholesterol were presented in Figure 6. Similar to what was observed in the second derivative spectra of amitriptyline in the pure lipids, the second derivative spectra in the lipids with cholesterol exhibited a bathochromic shift and increased in the derivative intensity of the minima. The Kp values were obtained using the data from the second derivative spectra, at a highest wavelength λmax in the absorption spectra (209 nm). The values of Kp were then calculated by fitting experimental data (∆D vs. [lipid]) to Equation (3) at 8 different lipid concentrations. The Kp values obtained were listed in Table 1 for DOPC, SOPC and DSPC and these lipids containing 28 mol% cholesterol. DOPC + CHOLESTEROL
Figure 6: Second derivative spectra of amitriptyline calculated from the absorption
spectra of Figure 4. Table 1: Partition coefficients (Kp) of amitriptyline at concentration 0.0225 mM into
the pure lipids DOPC, SOPC and DSPC and these lipids with 28 mol% cholesterol.
Kp values*
0 mol% cholesterol
28 mol% cholesterol
*The values reported were the mean and standard deviation of at least three independent measurements. As seen in Table 1, the Kp values of amitriptyline in the lipids DOPC, SOPC, DSPC followed the order: DOPC > SOPC > DSPC. It indicated that the partitioning of amitriptyline into the unsaturated lipid (DOPC and SOPC) was greater than that of the saturated lipid (DSPC). Possessing the cis-double bond, DOPC and SOPC molecules occupy more area ( 75 Å2/DOPC molecule, 65.5 Å2/SOPC molecule , respectively) than the saturated DSPC molecules ( 50-60 Å2/DSPC molecule) (see Figure 1). As a result, the more loosely packed DOPC and SOPC vesicles allow amitriptyline to partition more easily as compared to the more lightly packed DSPC vesicles. In addition, the experiments was carried out at 37 oC that was below the main phase transition of DSPC (Tm = 55 oC) and above the main phase transition of SOPC (Tm = 6 oC), DOPC (Tm = -17 oC). Since the physical state of lipid was determined by the transition temperature, DOPC and SOPC were in liquid – crystalline state, characterized by the high mobility because the acyl chains are more disordered whereas DSPC was in the solid-gel state with less mobility and more ordered acyl chains. The weak packability and high fluidity of DOPC and SOPC facilitated amitriptyline partition more effectively into these lipids relative to DSPC. This order for the partition of amitriptyline into DOPC, SOPC and DSPC is in agreement with the previous study, carried out on the partition of haloperidol into In the presence of 28 mol% cholesterol, the partition coefficient of amitriptyline into the saturated lipid DSPC and the mixed-chain lipid SOPC increased about 42% and 43%, respectively. In the unsaturated DOPC, however, the partition coefficient of amitriptyline decreased around 49%. This significant effect of cholesterol on the partitioning of amitriptyline into the lipid vesicles could be directly related to the interaction between cholesterol and the lipid vesicles. The ordering effect of cholesterol has been known to cause gel-state lipids become more disordered (i.e. fluidizing effect) and liquid-state lipids become more ordered. In the presence of cholesterol, the more ordered DOPC acyl chains resulted in a more tightly packed vesicles, reducing amitriptyline partition into the lipid vesicles. In DSPC vesicles, however, cholesterol fluidizes the gel-state lipid which allowed more amitriptyline penetrate into. In SOPC vesicles, the ordering effect should be expected since SOPC stays in the liquid state at 37oC. However, the partition coefficient of amitriptyline in SOPC did increase in the presence of cholesterol. It could be explained that, for the mixed-chain phospholipid SOPC containing one saturated chain - the sn1 and one acyl chain containing a double bond – the sn2 (see Figure 1), a combination of the ordering effect on the chain sn2 and the fluidizing effect on the chain sn1. The increase on the partitioning of amitriptyline into SOPC may be caused by the stronger disordering effect onthe chain sn1 that similar to the fluidity characterof CONCLUSION
In summary, it was indicated that the weak packability and high liquidity of DOPC and SOPC allowed amitriptyline partition more effectively as compared to DSPC. However, in the presence of cholesterol, the stronger fluidizing effect was induced on saturated DSPC and SOPC while the ordering effect was pronounced on the unsaturated phospholipids DOPC. As a consequence, cholesterol facilitated the partitioning of amitriptyline in DSPC and SOPC but inhibited the partitioning of amitriptyline in DOPC. These results support for the hypothesis, that is cholesterol has a significant effect on the partitioning of amitriptyline into the lipid membranes with different unsaturation degrees. In particular, the fluidizing and ordering effect of cholesterol on the partitioning of drug into the SOPC appears to be an important and interesting issue, which should be further studied. REFERENCES
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